The transcription factor CREB links a wide variety of extracellular signals to the activation of gene expression. Consequently, this factor has a prominent role in endocrine function. The appeal of the CREB pathway for study stems not only from its importance in diverse models of biological signaling, but also from the wealth of reagents that have been developed to monitor CREB function. As a result, this pathway provided some of the first insights into the control of gene expression by protein phosphorylation, one of the central themes in transcriptional regulation. The most widely held model is that CREB binds constitutively to a DMA element termed the cAMP-regulated enhancer (CRE), found in a large number of gene promoters. Gene activation is mediated by CREB phosphorylation, which allows recruitment of the coactivators, CBP and p300. Thus, which genes become activated is determined by the presence or absence of a CREB binding site in the promoter and activation perse is mediated by the functions of CBP/p300. Work supported by this grant during the past funding period challenges the dogma that CREB binds to its target sequences constitutively. It also supported our development of a novel method to establish not only which gene promoters are capable of interacting with CREB (i.e., the CREB regulon) but also whether qualitative or quantitative differences in signaling direct CREB to specific genomic targets. Termed SACO (Serial Analysis of Chromatin Occupancy), our approach combines chromatin immunoprecipitation with a modification of Long SAGE, a technique originally developed to analyze mixtures of RNA. The underpinning of the SACO technique is that a 21 nucleotide sequence generated from immunoprecipitated chromatin (termed genomic signature tags, or GSTs) can be localized to a unique position within a mammalian genome with 75% certainty. Thus, by collecting, sequencing, and localizing these GSTs, we can identify (and quantify) transcription factor binding sites in an unbiased manner. Our goals over the next funding period are to optimize the SACO methodology to make it more readily applicable, to develop bioinformatic approaches for motif analysis, to elucidate the mechanisms underlying the specific responses of CREB target genes to particular signaling pathways, to characterize the biological significance of bidirectional and reiterated CREs, and to characterize the functions of noncoding transcripts under CREB control.